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Domain-structure analysis of recombinant rat hormone-sensitive lipase

Österlund, Torben LU ; Danielsson, Birgitta LU ; Degerman, Eva LU ; Contreras, Juan Antonio LU ; Edgren, Gudrun; Davis, Richard C; Schotz, Michael C and Holm, Cecilia LU (1996) In Biochemical Journal 319(Pt 2). p.411-420
Abstract
Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module... (More)
Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains. (Less)
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author
organization
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Contribution to journal
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published
subject
in
Biochemical Journal
volume
319
issue
Pt 2
pages
411 - 420
publisher
Portland Press Limited
external identifiers
  • pmid:8912675
  • scopus:0029806454
ISSN
0264-6021
language
English
LU publication?
yes
id
3bb7f944-ec9c-440f-b8dd-c3658b1abb7d (old id 1109898)
alternative location
http://www.biochemj.org/bj/319/bj3190411.htm
date added to LUP
2008-07-28 11:09:20
date last changed
2017-07-30 04:27:07
@article{3bb7f944-ec9c-440f-b8dd-c3658b1abb7d,
  abstract     = {Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the alpha/beta-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and alpha/beta-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.},
  author       = {Österlund, Torben and Danielsson, Birgitta and Degerman, Eva and Contreras, Juan Antonio and Edgren, Gudrun and Davis, Richard C and Schotz, Michael C and Holm, Cecilia},
  issn         = {0264-6021},
  language     = {eng},
  number       = {Pt 2},
  pages        = {411--420},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Domain-structure analysis of recombinant rat hormone-sensitive lipase},
  volume       = {319},
  year         = {1996},
}